After blocking, 100 mL of ANG gel-filtration fractions containing 1% HSA and 0.05% Tween 20 were incubated for 1 h at RT. The wells were then washed with PBS containing 0.05% Tween 20 and incubated with 100 mL of rabbit anti-human ANG diluted in PBST at a final concentration of 10 mg/mL. ANG was revealed using peroxidase-conjugated Axitinib order goat anti-rabbit IgG (Sigma�CAldrich) and developed with 2,20-azino-bis(3-ethylbenzothi-azoline-6-sulfonic acid) (ABTS) (Sigma�CAldrich) and hydrogen peroxide as the substrate. The results of ANG determination in the gel-filtration fractions were reported in optical density (OD). Purification of human ANG�CIgM Serum samples donated by normal human volunteers were collected through the Blood Transfusion Service of National Institute of Rehabilitation.

Human IgM was purified in an IgM affinity column (HiTrap IgM Purification HP, Amersham Pharmacia Biotech, NJ, USA) and AKTAFPLC (Amersham Pharmacia Biotech). Procedures were carried out according to the manufacturer��s instructions. Briefly, serum samples were first prepared with ammonium sulfate until the final concentration was 0.8 M. The prepared sera were applied to the affinity column which had been pre-calibrated. IgM affinity binding buffer (20 nM sodium phosphate and 0.8 M (NH4)2SO4, pH 7.5) was then applied to wash out unbound factors; bound IgM was eluted by 20 nM sodium phosphate, pH 7.5. The flow rate of the overall purification procedure was 1 mL/min. Affinity of ANG�CIgM Immunoabsorbent columns were prepared with the antigens of interest coupled with cyanogen bromide-activated Sepharose (Pharmacia Biotech).

49 Two milligrams of protein were used for coupling with 1.5 mL CNBr-activated Sepharose. One gram of IVIg in 100 mL of PBS was loaded on the immunoadsorbent columm and run twice on the column at a speed of 1 mL/min at RT, followed by washing with PBS until the absorbance of the flow-through at 280 nm reached baseline values. Bound antibodies were eluted using a glycine�CHCl (0.1 M) buffer, pH 2.8, and 2 M NaCl followed by PBS and then diethanolamine (0.1 M) buffer, pH 11, and 2 M NaCl. The eluates obtained at different pH levels were brought to pH 7.0 and pooled. Two milliliters of the flow-through fractions were allowed to run through the sorbents for two more cycles and then used as effluent fractions. Eluates and effluents were dialyzed against PBS.

Quantification of total IgM Total IgM concentration was determined by nephelometry (Behring Nephelometer Analyzer, Marburg, Germany), according to standard procedures. Total ANG Entinostat and VEGF measurement The concentrations of total ANG and VEGF were measured by using commercially available ELISA kits (Quantikine Human Angiogenic Factor Immunoassay, R&D Systems, Minneapolis, MS, USA) according to the manufacturer��s protocol. Histology At the National Institute of Rehabilitation, malignant pathologic study is a routine part of evaluating osteosarcoma.